1. Field of the Invention
The present invention relates to a detecting reagent for an antiplatelet antibody. This detecting reagent is used to detect an antiplatelet antibody present in a blood of a patient at the time of, e.g., platelet transfusion.
2. Description of the Related Art
Platelets are akaryocites which are present in a blood and each of which has a diameter of 2 to 3 .mu.m. The platelets are produced by fragmentation of megakaryocytes present in the bone marrow. When platelet in a blood are brought into contact with damaged hemangioendothelial cells, they agglutinate with each other and play an important role for hemostasis.
In recent years, a platelet transfusion therapy has been made to aim at hemostasis and hemorrhagic prevention for patients who have a hemorrhagic tendency caused by thrombocytopenia or thrombopathy. By this therapy, many lot of patients are saved from death caused by hemorrhage, and their lives can be saved or prolonged. The amount of a platelet preparation used has been abruptly increased, with development of platelet transfusion.
An antibody against the antigen present on a surface membrane of the transfused platelet is produced in case that a large amount of platelets must be frequently transfused over a long period of time. Examples of such case is the platelet transfusion therapy for patients who suffer from acute leukemia and asplastic anemia. When this occurs, platelets transfused from random donors cannot cause any desired response of the patients, and no increase in the number of platelets is found after platelet transfusion. When platelet transfusion does not function well due to the reason described above, platelets having an antigen type which does not react with an antiplatelet antibody of such a patient must be transfused to this patient.
Antiplatelet antibodies are classified into an alloantibody and an autoantibody.
The antiplatelet alloantibody is produced by the above-mentioned platelet transfusion or pregnancy. The isoantibody does not only influence an effect of platelet transfusion described above but also causes post-transfusion purpura and neonatal thrombocytopenic purpura.
On the other hand, the antiplatelet autoantibody is produced in a patient who suffers from an autoimmune disease such as idiopathic thrombocytopenic purpura. The autoantibody is deemed to be a substance which causes an autoimmune disease.
Various conventional methods are available to detect the antiplatelet antibody as follows:
(1) Agglutination Assay
This method is the first method developed to detect the antiplatelet antibody and is originally used to detect a P1.sup.A antigen series and a Ko antigen series. According to this method, an IgM antibody is mainly detected, but an IgG antibody is rarely detected. This method tends to cause nonspecific agglutination caused by natural agglutination of platelets and has a low sensitivity.
(2) Complement-Fixation Reaction
After a sample serum, platelets, and a complement are reacted with each other, hemolysin labeled sheep blood cell are added to the reaction mixture, and an amount of consumption of the complement is determined from a degree of hemolysis of the hemolysin labeled sheep blood cell. This method can detect an HLA antigen on a surface membrane of the platelet and a blood group antigen of ABO type, but has a low sensitivity. In addition, it is difficult to detect the antiplatelet antibody if a sample serum has anticomplement activity.
(3) Antiglobuline Consumption Test
After platelets are reacted with an inactivated sample serum, the reaction mixture is washed. After an antiglobulin serum is added to the mixture, the mixture is subjected to a centrifugal operation. An anti-D antibody labeled blood cell is added to and reacted with the supernatant of the reaction mixture to detect an antiplatelet antibody in accordance with an antiglobulin titer of the supernatant. This method tends to cause pseudopositive determination and has a low sensitivity.
(4) RIA (Radioimmuneoassay)
The RIA is an antiglobulin test using an anti-human IgG labeled with a radioactive isotope. The isotope-labeled anti-human IgG is coupled to an antiplatelet antibody of a platelet of a patient, and the radioactive isotope elements coupled to the platelets are counted.
(5) MPHA (Mixed passive haemagglutination)
Platelets of solid phase are formed on the well wall of a U-shaped microplate, and a sample serum is added to and reacted with the platelets. After the well is washed, anti-human IgG labeled sheep red cell is added to the reaction mixture and is reacted with it. An agglutination pattern in which the sheep red cells collect in a central portion of the well is determined to be negative, and an agglutination pattern in which sheep red cells are scattered on the entire area is determined to be positive, thereby detecting a platelet antibody.
Of the conventional methods described above, the agglutination assay (1) requires only one step for the entire reaction and has simple test procedures. The agglutination assay, however, has disadvantages in that the IgG class antibodies cannot be detected and nonspecific agglutination tends to occur. Therefore, the agglutination assay is not suitable for practical applications.
Other conventional methods, i.e., the complement-fixation reaction (2) to the MPHA (5) require two or more steps each in the measurement. Therefore, the measurement time is prolonged, and the measuring operations are complicated.